► Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and…
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▼ Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and circannual rhythms. In the last eight years, a whole new dimension to melatonin’s role in biological organisms has emerged. In 1991 it was discovered [1,2] that melatonin exhibited antioxidant properties. Since then, many researchers [3,4] have found melatonin to be a powerful free radical scavenger and antioxidant. In the present study, the ability of melatonin to offer neuroprotection against glutamate, N-methyl-D-aspartate (NMDA), quinolinic acid (QA) and kainic acid (KA) (collectively referred to as the glutamate receptor agonists) was investigated. It was first shown that stress causes an increase in circulating glucocorticoid concentrations, which resulted in an increase the number of glutamate receptors on synaptic membranes in rat brain homogenate. Melatonin acted to reduce the number of glutamate receptors present on the synaptic membranes, implying that melatonin has neuroprotective properties, as overstimulation of the glutamate receptors leads to excitotoxicity and neurodegeneration. Further investigations showed that the glutamate receptor agonists induce neurodegeneration in primary neuronal cell cultures. Both co-treatment and posttreatment with melatonin against the glutamate receptor agonists, increased neuronal cell viability in a dose dependent manner. Melatonin also appeared to offer protection against quinolinic acid-induced neurodegeneration following intrahippocampal injections of quinolinic acid. The mechanism whereby melatonin offered this protection was investigated. The glutamate receptor agonists caused an increase in intracellular calcium concentrations, which is known [5] to be responsible for initiating the excitotoxic response. Melatonin had no effect on regulating intracellular calcium concentrations Additional studies indicated that melatonin was effective at scavenging superoxide radicals. Production of superoxide radicals was induced by the glutamate receptor agonists in primary neuronal cultures. Superoxide radicals induce lipid peroxidation, which involves the destruction of lipid membranes by chain reactions. By acting as an antioxidant, melatonin was able to reduce quinolinic acid-induced lipid peroxidation in rat brain homogenate, in a dose dependent manner. Melatonin was also effective at reducing lipid peroxidation induced by the glutamate receptor agonists in primary neuronal cultures. Melatonin therefore appeared to be offering neuroprotection by removing superoxide radicals and inhibiting lipid peroxidation. It had been reported [6] that melatonin inhibits nitric oxide synthase activity. This enzyme produces the free radical, nitric oxide, and can also produce superoxide radicals. Melatonin was able to reduce nitric oxide synthase activity in a dose dependent manner. This is a novel method of neuroprotection, as melatonin was now acting as an enzyme regulator. The results obtained demonstrate…

► Shift work is associated with early death, several types of cancer, and cardiovascular, metabolic, reproductive, and gastrointestinal disorders. Shift work’s disruption of circadian rhythms causes…
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▼ Shift work is associated with early death, several
types of cancer, and cardiovascular, metabolic, reproductive, and
gastrointestinal disorders. Shift work’s disruption of circadian
rhythms causes disease. While the increased health risks of
long-term shift work are recognized, studies of shorter periods of
shift work and rotating shift schedules are less conclusive.
Rotating shift schedules are used extensively in emergency
services, healthcare, hospitality, manufacturing, and
transportation. Shift work schedules affect workers’ performance,
and an individual’s perceived adaptation to their shift schedule is
associated with improved performance. With more than 100,000 deaths
per year in United States hospitals caused by medical error, work
performance of nurses is important.More than half of all American
nurses work rotating night shifts (at least three nights/month plus
days and evenings in that month). Nurses are routinely scheduled to
work only one or two night shifts, followed by days off. However
most rotating shift work studies focus only on the second – fifth
night shifts in a series; the current study investigated the
effects of a first night shift. After nurses had worked either a
night or day shift, saliva melatonin and cortisol levels were
assayed and perceived adaptation was rated, before sleep and on
awakening. Assays and ratings were then repeated after each nurse
worked the opposite-time shift. This study is also the first to
compare the same individuals’ results for these three measures
after both night shift and day shifts.Compared to working a day
shift, a single night shift lowered melatonin levels on awakening.
Cortisol was also lower on awakening, and higher before sleep;
perceived adaptation was rated worse. Individual assessments of
each participant’s results for melatonin, cortisol, and adaptation
suggest that the three measures are associated: individuals whose
melatonin and cortisol levels were more disrupted by working a
night shift also perceived that their adaptation to the night shift
schedule was worse. The specific changes in melatonin and cortisol
that occurred after a single night shift can be acutely
pathological even in healthy individuals—for example, a high
pre-sleep cortisol level prevents normal cellular immune response,
increasing susceptibility to most infections including the flu.This
study is unique in assessing melatonin and cortisol with perceived
adaptation after only one night shift. Working a single night shift
significantly disrupted melatonin and cortisol levels and rhythm,
and worsened adaptation. Physiological changes that in the
long-term increase disease risks also occur after a single night
shift—a health concern for rotating shift workers. Perceived
adaptation was worse after a single night shift, a potential safety
concern for workers and others who could be affected, including
hospital patients and drivers sharing the road.
Advisors/Committee Members: Novak, Colleen (Advisor).

►Melatonin is a hormone controlling the biorhythm of mammals and might be an indicator for harmony between individuals and their environment. The major ‘zeitgeber’ for…
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▼Melatonin is a hormone controlling the biorhythm of mammals and might be an indicator for harmony between individuals and their environment.
The major ‘zeitgeber’ for the release of melatonin by the pineal gland, regulated by the hypothalamus, is environmental light. It synchronizes the melatonin secretion with the 24- hour day/night cycle. Normally, melatonin levels are high in the night and low during the day.
There is a lot of discussion about building “mega” pig stables in The Netherlands. In these discussions animal welfare plays a big role. Since pig breeding is more and more intensified, several adaptations to the way of pig stabling are introduced, including adapting the light regimen to feeding regimen.
Until now no objective parameter is available to measure animal welfare in mammals. Because melatonin could be a possible parameter to estimate the (non) adaptation to another light regime.
We formed two groups of six pigs. One group was kept under a single light phase regimen, for a minimum of eight hours of light per day. The other group was kept under a phased light regimen, three periods of three hours of light spread over 24 hours. Each pig was sampled during a 24 hour period, by a saliva sample taken every hour.
In both groups no nocturnal rise in melatonin concentration was found. Both groups show a higher (no significant) mean melatonin concentration during light periods. There was no significant rise or fall in melatonin concentration caused by the change of environmental light from light to darkness and from darkness to light. There was no correlation between rise in melatonin concentration and food intake.
We could conclude we did not find a difference in melatonin release pattern between pigs kept under a discontinuous multiple light regimen and pigs kept under a continuous light regimen. If melatonin could be a useful parameter for measuring animal welfare cannot be confirmed by this experiment.
Advisors/Committee Members: van Geijlswijk, I.M..

►Melatonin, a hormone of the pineal gland, was evaluated in a variety of animal models of depression. Measurements of the frog righting reflex and rat…
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▼Melatonin, a hormone of the pineal gland, was evaluated in a variety of animal models of depression. Measurements of the frog righting reflex and rat locomotor activity showed that low doses of melatonin have a serotonin-like potentiating effect following monoamine oxidase inhibition. High doses of melatonin caused a reduction in the duration of rat immobility in the Porsolt model of depression and exerted a chlorpromazine-like effect on conditioned avoidance behaviour. In view of the indoleamine hypothesis of depressive disorders, the possibility of melatonin being a potential antidepressive is discussed and it is concluded that melatonin might be useful in the treatment of "agitated" depressions

► Introduction: Myocardial ischaemia and concomitant cell damage are caused by a reduction in the blood supply to the heart. To date, the most effective strategy…
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▼ Introduction: Myocardial ischaemia and concomitant cell damage are caused by a reduction in the blood supply to the heart. To date, the most effective strategy to salvage the myocardium is timely reperfusion which is unfortunately associated with further tissue damage. This phenomenon, termed ischaemia reperfusion injury, is associated with mitochondrial structural damage which could lead to death of cells previously damaged by ischaemia. Damaged and dysfunctional mitochondria play a key role in mediating tissue damage in this setting, thus the swift yet selective removal of these damaged organelles by mitochondrial autophagy – mitophagy could be of importance in cell survival and therefore is a potential therapeutic target.
Studies have shown that upregulation of autophagy during ischaemia/reperfusion is cardioprotective, however, very little is known about the role of mitophagy in this setting. Subsequently, the aims of this study were to (i) characterise the effect of ischaemia/reperfusion on functional recovery during reperfusion and to correlate this with mitochondrial oxidative phosphorylation capacity, infarct size and mitophagy in the working heart model using male Wistar rats; (ii) evaluate the effect of mitophagy manipulation on cardioprotection using the parameters listed above. To achieve this, used was made of melatonin, the pineal hormone, which is well-known for its cardioprotective effects.
Methods:
Male Wistar rat hearts were perfused ex vivo in the working mode using Krebs-Henseleit buffer and glucose (10mM) as substrate. After a stabilization period of 30 min, hearts were subjected to 20min global ischaemia followed by 30min reperfusion during which time functional recovery was monitored. Mitochondria were isolated from hearts at different times during the perfusion protocol: after stabilization for 30min, after 20min global ischaemia and after 30min of reperfusion. The mitochondrial pellets were used for measurement of mitochondrial oxidative phosphorylation using an Oxygraph as well as for western blotting to evaluate a number of indicators of mitophagy. In addition, hearts were subjected to the perfusion protocol as described above and freeze-clamped at the same time intervals for subsequent Western blotting for mitophagy markers in the cytosolic fraction. In a separate series melatonin (0.3, 50M) was added to the perfusate for 10min before and 10 min after ischaemia and the same parameters evaluated as above. For evaluation of infarct size by the tetrazolium method, hearts were stabilized for 30min, followed by 35min of regional ischaemia and 60min reperfusion.
Results:
Exposure of hearts to either 35min regional ischaemia/ 60min reperfusion or 20min global ischaemia/ 30min reperfusion was associated with impaired recovery of myocardial function during reperfusion, characterized by significant reduction in several haemodynamic endpoints including coronary flow, aortic output and total work performed. Exposure to 20min global ischaemia per se had no effect on mitochondrial oxidative…
Advisors/Committee Members: Lochner, Amanda, Salie, Ruduwaan, Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology..

▼ Autism spectrum disorder (ASD) is characterized by impairment in social interaction,
language impairment and repetitive behavior with varying degrees of severity. ASD
represents the lower end on a continuously distributed measure of autistic-like traits
(ALTs). Although a strong genetic component has repeatedly been identified in ASD,
the genetic cause of ASD is still unknown for the majority of ASD cases.
One of the main interests in this thesis is the neurobiology of melatonin, this
interest is based on findings indicating lower levels of melatonin in children with
ASD. In our investigations of rare mutations in melatonin related genes in subjects
with ASD, we identified a previously reported mutation that has been shown to
decrease the activity of one of the enzymes involved in the melatonin synthesis: the
acetylserotonin O-methyltransferase (ASMT) (paper I). In the analysis of five common
variations in the ASMT gene in relation to ALTs in the general population we found
association between a single nucleotide polymorphism and social interaction
impairment in girls (paper II).
To broaden the analysis of genetic influences on ALTs, we have performed
association analyses between ALTs in the general population and common variation in
genes previously found to be associated with ASD (RELN, CNTNAP2, SHANK3 and
CDH9/10 region) (paper III). Although these regions have previously been suggested
to be strong ASD candidate regions, our results do not suggest a major influence of the
investigated common variations on ALTs.
In the final paper, rare inherited genetic variations were investigated in a large
family with autism and language disorders. In this study, we used several techniques,
including whole exome sequencing and copy number variation analysis (paper IV). In
the family, several rare genetic variations which may partly explain the genetic etiology
for autism in this family were identified. We performed functional analyses for a
mutation identified in the CYP11A1 gene, indicating a gain of function mutation. The
CYP11A1 gene encodes the first enzyme in the steroid hormone biosynthesis, thus our
results may be in line with previous findings that have shown an elevated prenatal
steroidogenic activity in ASD.
In conclusion, we have identified both common and rare genetic variation that
may increase the genetic susceptibility for ASD. Our analyses have highlighted the
importance of taking both rare and common genetic susceptibility factors, as well as
different symptoms of the disorders, into account when elucidating the complex
inheritance of ASDs.

► During embryogenesis, vitamin A metabolism to retinoic acid (RA) is tightly regulated in a precise spatiotemporal pattern. Aberrant retinoid signalling in the wrong place or…
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▼ During embryogenesis, vitamin A metabolism to retinoic acid (RA) is tightly regulated in a precise spatiotemporal pattern. Aberrant retinoid signalling in the wrong place or at the wrong time has devastating consequences for development. Much is understood about the mechanisms by which at the level of specific tissues, RA synthesis from vitamin A is able to occur and activate target genes necessary for the developmental programme at any given stage. However, little is known about how retinoid transport and synthesis at the subcellular level occurs. Chapter 3 of this thesis investigates the subcellular compartments with which the vitamin A metabolising enzyme RALDH2 associates in embryonic neuronal cell lines. It is found that not only is RA synthesis tightly controlled by tissues, but that RA signalling within cells themselves may also be segmented. Aside from the importance of vitamin A during early development, it is now known that this dietary component is also vital in the maintenance of homeostasis during adulthood. This is particularly important for the adult CNS, where it is proposed that RA mediates hippocampal neurogenesis; hypothalamic neurogenesis; and physiological responses to seasonal changes in day length. The nocturnal hormone melatonin is a key signalling molecule relaying the length of the night to the rest of the brain and periphery, and by this means conveying information about the time of year. Vitamin A deficiency is known to severely reduce the amount of melatonin produced by the Quail pineal gland at night. Chapter 4 of this thesis investigates the circadian and circannual rhythms of retinoid genes such as the RALDH enzymes and retinoic acid receptors in both the SCN, and the pineal gland. RA is sufficient to increase the expression of the melatonin synthesising enzyme AANAT, and that this gene expression may be under the control of RALDH1.

Ransom, J. S. (2012). Compartmentalisation of retinoic acid synthesis in neuronal cells, and the role of RA in the control of melatonin synthesis by the pineal gland. (Doctoral Dissertation). University of Aberdeen. Retrieved from http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=196009 ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577574

Chicago Manual of Style (16th Edition):

Ransom, Jemma S. “Compartmentalisation of retinoic acid synthesis in neuronal cells, and the role of RA in the control of melatonin synthesis by the pineal gland.” 2012. Doctoral Dissertation, University of Aberdeen. Accessed May 25, 2019.
http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=196009 ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577574.

MLA Handbook (7th Edition):

Ransom, Jemma S. “Compartmentalisation of retinoic acid synthesis in neuronal cells, and the role of RA in the control of melatonin synthesis by the pineal gland.” 2012. Web. 25 May 2019.

Vancouver:

Ransom JS. Compartmentalisation of retinoic acid synthesis in neuronal cells, and the role of RA in the control of melatonin synthesis by the pineal gland. [Internet] [Doctoral dissertation]. University of Aberdeen; 2012. [cited 2019 May 25].
Available from: http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=196009 ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577574.

Council of Science Editors:

Ransom JS. Compartmentalisation of retinoic acid synthesis in neuronal cells, and the role of RA in the control of melatonin synthesis by the pineal gland. [Doctoral Dissertation]. University of Aberdeen; 2012. Available from: http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=196009 ; http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577574

►Melatonin is commonly used as a sleep-promoting supplement or an agent to relieve jet lag for its regulatory role in circadian rhythm of the central…
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▼Melatonin is commonly used as a sleep-promoting supplement or an agent to relieve jet lag for its regulatory role in circadian rhythm of the central nervous system. In addition, melatonin also functions as an important hormone in synchronizing activities of peripheral tissues mainly through MT1 and MT2melatonin receptors. Thus, developing subtype-selective melatonin receptor compounds to overcome the undesirable pharmaceutical properties of melatonin, such as non-selectivity and rapid elimination, would provide new treatment avenues for diseases associated with altered melatonergic system. With increasing evidence suggesting the involvement of MT2melatonin receptor in pathogenesis of diseases like depression, insomnia and type 2 diabetes, the development of MT2-specific selective ligands would facilitate the investigation of the pathogenic processes at the molecular level. Besides, current one-size-fits-all medications for these diseases need to be further improved, due to their several side effects and weak competency in controlling disease progression. Precise medicine, taking into account differences in an individual’s genetic composition, is thus proposed as an effective alternative to lower the risk of unfavorable outcome in treatment process. Therefore, this study investigated the effect of a highly selective MT2-specific compound 7b on five MT2 receptor variants (A52T, A74T, R138C, L166I, R222H) that are related to type 2 diabetes to support the notion of developing selective melatonergic ligands based on precise medicine strategy. The results of agonist-induced Ca2+ mobilization, ERK phosphorylation and cAMP accumulation displayed a decreased response from variants, compared to that from wild type. Interestingly, the response from 7b-stimulated MT2 receptor variants was higher than that from melatonin-induced. Further investigations pertaining to the effect of melatonin on glucose-induced insulin secretion from pancreatic β cells tended to support the possibility of applying melatonergic ligands in type 2 diabetes treatments. Furthermore, a clear map between MT2 receptor variants with different diseases, such as depression, insomnia, and type 2 diabetes, is required to construct a sound precision medicine system targeting the MT2melatonin receptor.

Insomnia in pregnancy is associated with depression, preeclampsia, gestational diabetes, and preterm labor. In normal pregnancies, maternal plasma melatonin levels increase significantly as pregnancy proceeds, reaching its peak near term. However, the role of melatonin in insomnia during pregnancy is not known. The objective of this study was to measure nocturnal saliva melatonin levels in pregnant women with and without insomnia. Results did not show a significant difference in melatonin levels between insomniac (treated and untreated) and healthy pregnant women in all trimesters. However, sub-group analysis showed significantly lower melatonin levels in untreated insomniac pregnancies compared to healthy pregnancies and those treated with sleep medications. Results of this study confirmed lower levels of nocturnal melatonin in untreated pregnancies with insomnia. Future research is needed to investigate the safety and efficacy of melatonin supplementation for the treatment of insomnia in pregnancy, replacing psychotropic drugs.

► The circadian system provides an integrating mechanism for synchronization of biological processes with the regular 24-hour light and dark changes in the environment. In some…
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▼ The circadian system provides an integrating mechanism for synchronization of biological processes with the regular 24-hour light and dark changes in the environment. In some teleost species, ocular melatonin levels exhibit a circadian periodicity with elevated levels during the dark as compared to light, thereby regulating the circadian rhythms of several biological functions, such as the diurnal suite of events that help the retina anticipate changes in ambient light. To gain a better understanding of the diurnal variation in gene expression, I analyzed the changes in gene expression in the eye of zebrafish. Dual color oligonucleotide microarrays were used to compare total RNA harvested from eyes of adult zebrafish at midday and midnight. Statistical analyses identified 44 genes which showed significant, 2-fold or more change; 26 genes showed decreased expression at midnight (D/L ≤ 0.5) and 18 genes showed increased expression at midnight (D/L ≥ 2). Seven genes were further analyzed using qPCR. The results of qPCR identified AANAT, Mel1a1, Mel1a3, Mel1b1, Mel1b2 and Melc as genes that showed significant change in expression at dawn, dusk, midday and midnight. These results suggest that expression of melatonin receptors is subject to diurnal regulation.
Advisors/Committee Members: García, Dana M. (advisor), Koke, Joseph R. (committee member), Dharmasiri, Nihal (committee member).

►Melatonin, a powerful antioxidant, offers potential human benefits in the fields of medicine, nutrition, and food science. While best understood in a mammalian system, melatonin…
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▼Melatonin, a powerful antioxidant, offers potential human benefits in the fields
of medicine, nutrition, and food science. While best understood in a
mammalian system, melatonin has been identified in plants and dietary
melatonin has been shown to increase circulating levels in the blood. Thus,
there exists a great interest in extracting and detecting melatonin present in
edible plant matrices. Extraction techniques such as liquid/liquid, solid phase,
and solid/liquid extraction were investigated and compared to determine the
best approach for isolating melatonin from fruit. Enzyme-linked immunoassay
(ELISA), fluorescence, and mass spectrometry were investigated for their use
as detection methods for melatonin originating in fruit systems. Additionally,
the stability of melatonin in a pH 3.5 buffered model system was studied to
gain preliminary information regarding melatonin heat and light stability. It was
determined that melatonin is both heat stable and light stable for up to one
hour (85C and 17 par, investigated separately). Solid/liquid extraction using
ethyl acetate as a solvent was determined to be the best extraction procedure
while high performance liquid chromatography-mass spectrometry with the
use of a deuterated internal standard was the preferred detection method. A
significant amount of work remains in the area of quantification of melatonin
from fruit systems.

► Vocalization is a prominent feature of social communication among vertebrates. For energetically costly vocal-acoustic courtship behaviors, timing across seconds, days, and seasons is critical and…
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▼ Vocalization is a prominent feature of social communication among vertebrates. For energetically costly vocal-acoustic courtship behaviors, timing across seconds, days, and seasons is critical and can enhance sender-receiver coupling, reproductive success, and reproductive isolation. Many species of fish produce sound to communicate in different social contexts, such as courtship. Here, I investigated hormonal, neuronal, and genetic mechanisms underlying the timing of vocal behavior in the plainfin midshipman fish (Porichthys notatus), across timescales spanning milliseconds to seasons. I demonstrated that the robust daily rhythm of midshipman male's nocturnal courtship vocalization is under endogenous, circadian control. Exogenous delivery of melatonin, the nocturnal hormone in vertebrates, rescued the inhibition of courtship vocalization under constant light, which abolishes endogenous melatonin production. Melatonin also rescued the inhibition of neural excitability in the midshipman vocal network under constant light. Furthermore, melatonin receptor 1b mRNA was shown to be expressed in neuroendocrine, sensory (including auditory) and vocal motor pathways. Together, these results support the hypothesis that melatonin plays a central role in timing the nocturnal midshipman courtship vocalization by acting on specific neural pathways. Finally, I used RNA-sequencing to characterize the transcriptome of the vocal motor nucleus (VMN), the final node of the hindbrain vocal pattern generator that directly determines vocalization temporal characteristics such as duration and frequency. I identified a suite of candidate genes, including ion channels, for shaping the precise and synchronous firing of VMN motor neurons. Many candidate genes showed day-night and seasonal changes in expression. Furthermore, enrichment and high expression of cellular respiration genes in VMN compared to the surrounding hindbrain tissue likely enable midshipman courtship calls that can last up to hours, and suggest that the neural patterning of vocal behavior is energetically costly. Finally, high expression of several antioxidant genes in VMN suggested a high capacity for combating cellular respiration-generated oxidative stress, which may also enable long duration courtship call production. Altogether, these chapters identify mechanisms underlying the timing of vocalization that may be applicable across other lineages of vertebrates, including birds and mammals, which exhibit rhythmic production of vocalization across multiple timescales.
Advisors/Committee Members: Fetcho,Joseph R. (committeeMember), Deitcher,David Lawrence (committeeMember), Place,Ned J. (committeeMember).

► The pineal gland is a naturally calcifying endocrine organ which produces and releases the sleep-promoting hormone melatonin which also serves as an antioxidant. Fluoride is…
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▼ The pineal gland is a naturally calcifying endocrine
organ which produces and releases the sleep-promoting hormone
melatonin which also serves as an antioxidant. Fluoride is
attracted to the calcium in the pineal gland and inhibits the
synthesis and activity of melatonin, induces oxidative stress, and
causes cellular changes to the neurons of the hippocampus.
Morphological changes to the pineal gland have been demonstrated
with increased age, exposure to light during a melatonin production
period, or sleep deprivation. This study sought to examine the
effects of fluoridated water on the morphology of the pineal gland.
The effects of a fluoride-free flush were compared to fluoride
treatment. Group 1, previously raised on fluoridated tap water
served as a control that was sacrificed at the onset of the
experiment. The remaining four groups were then subjected to a
four-week fluoride-free diet with Group 2 being sacrificed at the
end of this period. Group 3 was maintained on fluoride-free food
and water while Groups 4 and 5 were switched to fluoridated water
for the remaining four weeks. The fluoride-free flush resulted in
an increase in the number of supporting cells and pinealocytes and
a decrease in the nuclear diameter of pinealocytes, suggesting that
the flush encouraged growth of the gland. Fluoride treatment had no
effect on the number of supporting cells, but decreased the number
of pinealocytes and their nuclear diameter, suggesting that
fluoride is detrimental to the pineal gland.
Advisors/Committee Members: Womble, Mark (Advisor).

► The circadian rhythm is an internal body cadence, responsible for regulation of sleep in all mammals. In humans, this clock is altered by several factors,…
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▼ The circadian rhythm is an internal body cadence, responsible for regulation of sleep in all mammals. In humans, this clock is altered by several factors, including light and secretion of the hormone melatonin. Within the intensive care unit (ICU) population, it is well evidenced that patients suffer from circadian dysregulation, often for long periods of time. Additionally, many parallels have been noted between severely fragmented sleep and delirium, an acute neurological condition frequently observed in ICU patients. A prospective cohort pilot study of five subjects was undertaken to enable a greater understanding of both sleep in the ICU and the relationship between circadian rhythm and delirium. From a total of thirty-six urine samples per subject, excretion of 6-sulphatoxymelatonin (aMT6s), the urinary metabolite of melatonin was analyzed. T-test comparison (p=0.05) of mean aMT6s (ng/mL) revealed significant differences in the nighttime excretion between subjects in this study and healthy individuals. No significant differences were observed with t-test comparison of mean aMT6s of the first 24 hours from the current study to ICU subjects in previous literature. No subjects were identified as delirious in the study and therefore no relationship could be found between circadian rhythmicity, as evidenced by melatonin excretion and delirium in this study population.
Advisors/Committee Members: McMillan, Diana (Nursing) (supervisor), Diehl-Jones, William (Nursing) .

▼ doses under controlled conditions during the daytime, when endogenous levels are low. Study findings have demonstrated that exogenous melatonin improves sleep, increases peripheral heat loss, and decreases core body temperature (CBT). These thermoregulatory adjustments mimic those that occur around habitual bedtime, when endogenous melatonin levels are high. The emergences of artificial light and stimulants i.e., caffeine have impacted the behavior and physiology that normally precede sleep. Caffeine may independently impact sleep/wakefulness, or in conjunction with the thermoregulatory system. Bright light during the biological night suppresses melatonin and changes the thermoregulatory pattern that precedes nocturnal sleep; these changes may ultimately impact the sleep/wakefulness system. To improve our understanding of physiological mechanisms promoting and disrupting sleep/wakefulness, it is important to examine the connection between melatonin and the sleep/wakefulness and thermoregulatory systems, and the impact of environmental and behavioral factors on these systems. Therefore, the aims of this dissertation were to: 1) determine the effect of a melatonin receptor analogue ramelteon, on daytime sleep and body temperature, and the relationship between the two variables; 2) determine the effect of daytime exogenous melatonin on resting energy expenditure, (REE); and 3) determine the individual and compound effects of caffeine and bright light on thermoregulatory and sleep physiology at night.
Consistent with our hypotheses, 1) ramelteon significantly improved daytime sleep, lowered CBT, and increased peripheral heat loss 2) exogenous melatonin decreased REE during the daytime, and 3) caffeine delayed the nocturnal rise in peripheral heat loss, attenuated the fall in CBT, while the combination of caffeine and bright light decreased slow wave sleep and increased sleep onset latency.
These findings suggest that melatonin may play an important role in the regulation of sleep/wakefulness as evidenced by the effect of daytime ramelteon administration on sleep and thermoregulatory physiology and the effect of daytime exogenous melatonin on REE. Finally, caffeine and bright light had a negative impact on nocturnal sleep and these effects may be mediated in part by their impact on the thermoregulatory system.
Advisors/Committee Members: Robert S Mazzeo, Kenneth P Wright Jr, Christopher A Lowry.

► On the basis of clinical studies, some researchers have advocated that the neurohormone and antioxidant melatonin, shown to possess intrinsic anticancer properties, be used as…
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▼ On the basis of clinical studies, some researchers have advocated that the neurohormone and antioxidant melatonin, shown to possess intrinsic anticancer properties, be used as co-therapy in cancer patients being treated with the antineoplastic agent 5-fluorouracil, as increased patient survival times and enhanced quality of life have been observed. The focus of this research was thus to investigate the mechanisms of this seemingly beneficial drug interaction between 5-fluorouracil and melatonin. Metabolism studies were undertaken, in which it was established that there is no hepatic metabolic drug interaction between these agents by cytochrome P450, and that neither agent alters the activity of this enzyme system. Co-therapy with melatonin is thus unlikely to alter plasma levels of 5-fluorouracil by this mechanism. Novel mechanisms by which 5-fluorouracil is toxic were elucidated, such as the induction of lipid peroxidation, due to the formation of reactive oxygen species; decreases in brain serotonin, dopamine and norepinephrine levels, possibly leading to depression; hippocampal shrinkage and morphological alterations and lysis of hippocampal cells, which may underlie cognitive impairment; and a reduction in the nociceptive threshold when administered acutely. All these deleterious effects are attenuated by the co-administration of melatonin, suggesting that the agent exhibits antidepressive and analgesic properties, in addition to its known antioxidative and free radical-scavenging abilities. This suggests that melatonin cotherapy can significantly decrease 5-fluorouracil-induced toxicity, but this may also exert a protective effect on cancer cells and thus compromise the anticancer efficacy of 5-fluorouracil. It was, furthermore, found that stimulation of indoleamine 2,3-dioxygenase activity, mediated by increases in superoxide anion and interferon-γ levels, may underlie resistance to 5-fluorouracil therapy. Melatonin was shown to increase superoxide anion levels in vivo, and this is believed to be by conversion to the metabolite and known oxidant 6- hydroxymelatonin. This highlights that the possible deleterious effects of melatonin metabolites should be studied further. Serum corticosterone levels and cytokine profiles are unaltered by both 5-FU and melatonin, suggesting that these agents may be used by HIV infected individuals without promoting the progression to AIDS. It can thus be concluded that melatonin co-therapy is potentially useful in countering 5-fluorouracil toxicity.

Melatonin is a indolamine synthesized primarily by the pineal gland, whose function is associated with the marking of the dark phase. Beyond this chronobiotic function, melatonin also plays a role in defense and is synthesized by other sites. It may exert paracrine and autocrine action, like in immunocompetent cells. High substantially concentrations of melatonin are found in the cerebrospinal fluid (CSF) that has been linked to the synthesis of melatonin by the central nervous system cells (CNS), such as microglia. Knowing that these cells are the resident macrophages in the CNS and that melatonin synthesis by these phagocytes is proven, our study aims to assess whether cerebellar microglia synthesize this indolamine and whther this acts enhancing phagocytosis of these cells. Our results show that blocking the melatonin receptors with the antagonist, luzindole, both the exogenous melatonin-induced phagocytosis and the basal phagocytosis decreased, indicating that there is melatonin synthesis by cerebellar microglia which acts on phagocytosis. These results are significant and indicate that melatonin synthesized by microglia may be related to the neural environment homeostasis. In this way, our data can contribute, for example, in studies to establish new therapeutic strategies for neuroinflammatory diseases.

Malaria is the most killing parasitic disease in the world. Half of the world population is at risk of contracting the disease, which kills over 1 million people, being children under 5 the most affected. The fever periodicity is the characteristic symptom of the disease. The fever is a result of the burst of the erythrocyte when the parasite leaves the host cell to infect other ones. This event is highly synchronous, with the parasites going out of the cells at the same time. For this to happen, the cellular events that are necessary for parasite growth have to be very well regulated. The circadian hormone melatonin is the signal that synchronizes the intraerythrocytic cycle of Plasmodium. In this work, we report that this synchrony, observed in the majority of the parasites species, could be used as a way to evade the immune system, assuring the continuity of the infection. When we disrupt this synchrony with luzindole, a melatonin antagonist, we observe that a suboptimal dose of the antimalarial chloroquine increases the survival of the infected mice. We also report that P. berghei, rodent parasite that possess and unsynchronized infection, cant perceive the hormone. Unlike what is observed in species that have a synchronous infection, melatonin fail to induce intracellular calcium increase or promote cell cycle synchronization in vitro. Here we also report the construction of knockout vector for Plasmodium, to be…

T Lymphocytes are exposed to severe homeostatic regulation from development stage up to their maturation, clonal expansion and cell death. Endogenous or exogenous compounds altering the physiological functions of T lymphocytes can modulate important steps of the immune response. Tryptophan plays an important role for maintaining the homeostasis of T lymphocytes and is the precursor of the melatonin synthesis. In this study we evaluated the effects of tryptophan, melatonin and their oxidation products concerning the main activation and deactivation processes of T lymphocytes. Firstly, we analyzed the biological effects of L-kynurenine (KYN, a compound formed at the tryptophan metabolization pathway), melatonin and its oxidation products, N-acetyl-N-formyl-5-methoxykynuramine (AFMK) and N-acetyl-5- methoxykynuramine…

The melatonin synthesis by human mononuclear phagocytes starts after the induction by IgA opsonized or not zymosan. This production is dependent on the activation of NFKB pathway since the pharmacological block of the pathway by PDTC or ALLN reduces the melatonin concentration in culture supernatants. The NFKB localization temporally varies after initial stimulus and the presence of specific subunits in the cell nucleus is different in activated cells when compared with control cells. We observed the presence of p50 subunit in all experimental conditions (control, zymosan, opsonized zymosan), but the Rel A and c-Rel subunits were only detected in treated cells. Melatonin shows activity over immune cells in many experimental models, but the phagocytosis model was not yet reported in literature. We observed that melatonin (1 nM) is able to potentiate the non opsonized…

►Melatonin is rhythmically synthesized and released by the pineal gland and, in some species, retina during the night and regulates many physiological and behavioral processes…
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▼Melatonin is rhythmically synthesized and released by the pineal gland and, in some species, retina during the night and regulates many physiological and behavioral processes in birds and mammals. Chick diencephalic astrocytes express two melatonin receptor subtypes in vitro, and melatonin plays a role in regulating metabolic activity. We examined the role of glial cells in circadian function and asked if melatonin modulated glial functions within the retina and the brain. Calcium waves were potentiated by physiological concentrations of melatonin. Melatonin increased resting calcium levels and reduced gap junctional coupling among astrocytes at these same concentrations. Both mouse and chick diencephalic and telencephalic astrocytes express melatonin receptor protein. Nanomolar melatonin modulated astrocytic calcium waves of the mouse and chick diencephalon but not waves of the telencephalon. Mammalian intercellular calcium waves spread farther than avian calcium waves, and the nature of the spread of the waves differed between telencephalic and diencephalic mammalian astrocytes. These differences in propagation were abolished by melatonin. Using northern analysis, we identified period2, period3, cryptochrome1, cryptochrome2, clock, melanopsin and peropsin within chick diencephalic astrocytes. The clock genes cry1 and, per2 were expressed rhythmically in a LD cycle, but metabolic activity was not rhythmic. When cells were placed in constant darkness and rhythmically administrated melatonin, a robust rhythm in glucose uptake was induced without a coordinated clock gene rhythm, suggesting rhythmic clock gene expression and metabolic activity are separable processes. Melatonin affected visual function as assessed by electroretinogram. Circadian rhythms of a- and b-wave implicit times and amplitudes were observed. Melatonin (1 mg/kg and 100 ng/kg) decreased a- and b-wave amplitudes greater during the night than during the day and it increased a- and b-wave implicit times while 1 ng/kg melatonin had little to no effect over the saline controls. These data indicate that melatonin modulates glial intercellular communication, affects metabolic activity in astrocytes, and may play a role in regulating a day and night functional shift in the retina, at least partially through M??ller glial cells. Thus, melatonin can regulate glia function and thereby, affect outputs of the vertebrate biological clock.
Advisors/Committee Members: Cassone, Vincent M. (advisor), Bell-Pedersen, Deborah (committee member), Dryer, Stuart (committee member), Earnest, David (committee member), Zoran, Mark J. (committee member).

► Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and…
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▼ Acetaminophen and N,N-dimethylformamide (DMF) are compounds which are extremely toxic to the liver. Acetaminophen is a drug which is well known for its analgesic and antipyretic properties. However, the abuse potential of this agent as a non-narcotic analgesic in alcoholics is well known. It is also the leading cause of overdose in England. DMF toxicity results mainly from occupational exposure. At present there are no known reports of an antidote for DMF poisoning, while N-acetylcysteine, the antidote for acetaminophen poisoning, is known to produce adverse effects. The present study evaluates the potential of melatonin as an antidote for acetaminophen and DMF poisoning. This study also investigates the mechanism underlying acetaminophen addiction and abuse. Initial studies involved in vitro techniques in an attempt to remove the complexities of organ interactions. The photodegradation studies, using ultraviolet (UV) light, revealed that melatonin accelerates the rate of acetaminophen degradation in the presence of air, and reduces the rate of degradation in the presence of nitrogen. This study also revealed that melatonin is rapidly degraded in the presence of air, following UV irradiation. The effect of DMF on hydroxyl radical generation was also determined. DMF was shown to act as a free radical scavenger, rather that a generator of free radicals. The in vitro studies were followed by lipid peroxidation determination. DMF (0.4ml/kg and 0.8ml/kg) did not produce any significant increases in lipid peroxidation in the liver. Three different doses of acetaminophen (30mg/kg, 100mg/kg, and 500mg/kg) were administered to rats for seven days. Acetaminophen (500mg/kg) was shown to significantly increase (p<0.05) lipid peroxidation in the liver. Melatonin (2.5mg/kg) was not able to significantly reduce the damage. The lower doses of acetaminophen (30mg/kg and 100mg/kg) did not increase lipid peroxidation. Electron microscopy studies showed that DMF adversely affects the liver, and in particular, the endoplasmic reticulum. Co administration of melatonin (2.5mg/kg) was able to reduce the damage. Further experiments need to be performed before an accurate assessment can be made on the ability of melatonin as an antidote for DMF and acetaminophen poisoning. Several experiments were done in an attempt to uncover the biochemical mechanism underlying acetaminophen addiction and abuse. The first experiment targeted the liver enzyme tryptophan-2,3-dioxygenase (TDO). This enzyme is the major determinant of tryptophan levels in vivo. Acetaminophen administration (100mg/kg for three hours) was shown to significantly inhibit (p<0.05) the activity of TDO, indicating increased peripheral levels of tryptophan. This experiment was followed up with determination of brain serotonin and pineal melatonin. Brain serotonin was determined using the ELISA technique. Melatonin was estimated using this technique as well as with pineal organ culture. Acetaminophen administration (100mg/kg for three hours) significantly increased (p<0.05) brain…

► Carbon dots is a very novel nanomaterial with fluorescence property. It has multiple characteristic, such as : easily and facile synthesis, good water solubility, chemistry…
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▼ Carbon dots is a very novel nanomaterial with fluorescence property. It has multiple characteristic, such as : easily and facile synthesis, good water solubility, chemistry inert, lower toxic, surface functionalize and resisting photobleaching. Therefore, with all the superioritys, carbon dots can apply in many research regions, like : photocatalysis, photoluminescence, chemosensors, metal ions probes, biosensors, bioimaging even in the drug delivery system.
This thesis is about using polypyrrole functionalize carbon dots(PPy-Cdots) to detect melatonin. Polypyrrole is a conducting polymer, it has three main properties to functionalize it to our synthesis carbon dots. The first one is good biocompatible property that reducing the error from the environment. The second is well signal transferring. No matter what importing or exporting signals, it will not easily influence by functionalize agent itself. The third it can protect carbon dots from polluting and let the analysis result inaccurate. Melatonin is a hormone that is general presence in organisms. It can affect our biological rhyme, natural rest. It also has functions of anticancer and increased immunity. Recently, melatonin is already applied in curing many kinds of diseases, for instance: insomnia and depression. As a result, detecting the quantity of melatonin in human body is an important subject.
As a consequence, we synthesis a polypyrrole functionalize catbon dots, not only make the luminescence increased but also preventing interference from possible resources. Melatonin has similar absorbance region with our sample, so partial energy will absorb by analytes when sample excited by laser cause quenching effect happened. Later, we use this phenomenon to quantify the amount.
Advisors/Committee Members: Hui-Fen Wu (committee member), Fu-ken Liu (chair), Sarah Y. Chang (chair).

Background: Endometriosis is a benign condition that affects women in childbearing age. It is a estrogen-dependent disease, multifactorial, associated with a generalized inflammatory response in the peritoneal cavity, being the most common cause of chronic pelvic pain. Objective: This study have compared the effect of melatonin 10 mg / day with placebo in pain and in serum levels of brain-derived neurotrophic factor (BDNF) in patients with endometriosis. Methods: We conducted a randomized, double-blind, parallel, placebocontrolled trial. We included women at aged between 24 and 52 years with the diagnosis of endometriosis by laparoscopy selected from the daily schedule of consultations of the Gynecology outpatient clinic and by calling the local media, for the period September 2010 to April 2012. Questionnaires were used to evaluate the frequency and intensity of pain (during intercourse, urination and work), depressive symptoms, level of catastrophic thinking and the Structured Clinical Interview for DSM-IV (SCID) for psychiatric diagnoses. Results: In the analysis by intention to treat, the…

► Background: Shift work is associated with increased risk of cardiovascular disease and cancer, where decreased melatonin has been proposed as an intermediate in the causal…
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▼ Background: Shift work is associated with increased risk of cardiovascular disease and cancer, where decreased melatonin has been proposed as an intermediate in the causal pathway. The influence of physical activity on melatonin has rarely been studied in an observational setting, and it may be important in mediating the effects of shift work. We aimed to assess the influence of energy expended during physical activity of different intensities on melatonin among rotating shift nurses. We hypothesized that physical activity before the night shift would lessen the decrease in melatonin production that occurs with exposure to light at night.
Methods: 123 female rotating shift nurses working at Kingston General Hospital were recruited over a one-year period. Physical activity and sedentary behaviours for each participant were recorded during both a day and a night shift using activity diaries, and analysis was restricted to activities between 3 p.m. and 7 a.m. Concentrations of urinary 6-sulfatoxymelatonin, a melatonin metabolite, in morning void urine samples were analyzed for each shift.
Results: The average age of participants was 41 years, and 60% were overweight or obese (body mass index ≥ 25 kg/m2). An average of 6.9 and 5.2 hours of sleep were reported after the day shift and night shift, respectively. Sedentary behaviours such as standing and television watching accounted for over half of the total reported energy expenditure. During the day shift, energy expended in moderate and vigorous intensity physical activity between 3 p.m. and 7 a.m. was negatively associated with melatonin levels (p=0.024, R2 = 0.09). During the night shift, energy expended in sedentary behaviours was negatively associated with melatonin levels (p=0.008, R2 = 0.03).
Conclusions: Physical activity energy expenditure explains only a small amount of melatonin variation, suggesting that other factors are influencing melatonin production, or that melatonin production is minimally effected by these patterns of physical activity.